Determinants of specificity for alpha-conotoxin MII on alpha 3 beta 2 neuronal nicotinic receptors

The competitive antagonist alpha-conotoxin-MII (alpha-CTx-MII) is highly selective for the alpha 3 beta 2 neuronal nicotinic receptor. Other receptor subunit combinations (alpha 2 beta 2, alpha 4 beta 2, alpha 3 beta 4) are >200-fold less sensitive to blockade by this toxin. Using chimeric and mutant subunits, we identified amino acid residues of alpha 3 and beta 2 that participate in determination of alpha-CTx-MII sensitivity. Chimeric alpha subunits, constructed from the alpha 3 and alpha 4 subunits, as well as from the alpha 3 and alpha 2 subunits, were expressed in combination with the beta 2 subunit in Xenopus laevis oocytes. Chimeric beta subunits, formed from the beta 2 and beta 4 subunits, were expressed in combination with alpha 3. Determinants of alpha-CTx-MII sensitivity on alpha 3 were found to be within sequence segments 121-181 and 181-195. The 181-195 segment accounted for approximately half the difference in toxin sensitivity between receptors formed by alpha 2 and alpha 3. When this sequence of alpha 2 was replaced with the corresponding alpha 3 sequence, the resulting chimera formed receptors only 26-fold less sensitive to alpha-CTx-MII than alpha 3 beta 2. Site-directed mutagenesis within segment 181-195 demonstrated that Lys185 and Ile188 are critical in determination of sensitivity to toxin blockade. Determinants of alpha-CTx-MII sensitivity on beta 2 were mapped to sequence segments 1-54, 54-63, and 63-80. Site-directed mutagenesis within segment 54-63 of beta 2 demonstrated that Thr59 is important in determining alpha-CTx-MII sensitivity.